Methods and Compositions for the Treatment of Retinopathy and Other Ocular Diseases

ABSTRACT

Compositions and methods for the treatment of ocular diseases are disclosed.

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 62/093,848, filed on Dec. 18, 2014.The foregoing application is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of ocular diseases. Specifically,the invention provides novel compositions and methods for the treatmentof ocular diseases, particularly ocular diseases characterized byabnormal vascularization such as retinopathy.

BACKGROUND OF THE INVENTION

Eye disease is a significant cause of morbidity in the U.S. andthroughout the world. For example, retinopathy is one of the most commoncauses of vision loss in the world and age-related macular degenerationis the most common cause of blindness in people over 50 in the U.S.While therapies have improved for many eye diseases, there is still aneed for methods and compositions for inhibiting or treating eyediseases, particularly those characterized by aberrant vascularization.

SUMMARY OF THE INVENTION

In accordance with one aspect of the instant invention, methods fortreating, inhibiting (e.g., reducing), and/or preventing an oculardisease in a subject are provided. The methods comprise theadministration of at least one inhibitor of the induction or activity oftryptophan degradation and/or of the downstream pathways that respond tothis process. In a particular embodiment, the methods comprise theadministration of an inhibitor of IDO1.

Compositions for the treatment of an ocular disease are also provided.The compositions comprise at least one inhibitor of the induction oractivity of tryptophan degradation or of the downstream pathways thatrespond to this process (e.g., an inhibitor of IDO1) and at least onepharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides images of retinal flat mounts from wild-type (left) andIdo1 knockout (Ido^(−/−); right) mice at P17. FIG. 1B provides a graphof the neovascularization observed in the retina from wild-type (WT) andIdo1 knockout (Ido^(−/−)) mice at P17 (left) and a graph of theavascular region observed in the retina from wild-type (WT) and Ido1knockout (Ido^(−/−)) mice at P17 (right). Data are +/−SEM; N≧24/group;*** P<0.0001.

FIG. 2A provides a graph of the neovascularization observed in theretina from wild-type (WT), Ido1 knockout (Ido^(−/−)), IFN-γ knockout(Ifng^(−/−)), and double knockout (Ifng^(−/−) Ido^(−/−)) mice at P17.FIG. 2B provides a graph of the neovascularization observed in theretina from wild-type (WT), IL6 knockout (Il6^(−/−)), IFN-γ knockout(Ifng^(−/−)), and double knockout (Ifng^(−/−) Il6^(−/−)) mice at P17.

FIG. 3 provides representative images of isolectin staining of thevasculature in retinal flat mounts at P17 from WT and Ido1^(−/−)neonates maintained either under constant normoxia (left column) orexposed to hyperoxia from P7-P12 to trigger oxygen-induced retinopathy(OIR; middle column). Right column (OIR (magnified)) provides highermagnifications of the OIR images in the middle column.

FIG. 4A provides a graph of neovascular (NV) area over total retinalarea quantitatively assessed for isolectin stained retinal flat mountsprepared at P17 from WT, Ido1 knockout (Ido1^(−/−)), and Ido2 knockout(Ido2^(−/−)) neonates exposed to hyperoxia from P7-P12 to induce OIR(N≧16). Corresponding cohorts maintained under constant normoxia wereevaluated as a baseline comparison (N≧10). Data are means+/−SEM; ***P<0.001; ns, not significant. FIG. 4B provides a graph of NV area overtotal retinal area quantitatively assessed for isolectin stained retinalflat mounts prepared at P17 from WT and Ido1^(−/−) neonates of BALB/c orC57BL/6 strain background exposed to hyperoxia from P7-P12 to induce OIR(N≧14). Data are means+/−SEM; **** P<0.0001.

FIG. 5A provides a graph of vessel area over total retinal areaquantitatively assessed for isolectin stained retinal flat mountsprepared at P17 from WT and Ido1^(−/−) neonates maintained underconstant normoxia (N=10). Data are means+/−SEM; ns, not significant.FIG. 5B provides a graph of NV area over total retinal areaquantitatively assessed for isolectin stained retinal flat mountsprepared at P17 from WT and Ido1^(−/−) neonates exposed to hyperoxiafrom P7-P12 to induce OIR followed by intraocular injections ofinterferon-γ (IFNγ) neutralizing antibody or an isotype matched negativecontrol antibody (N≧9). Data are means+/−SEM; ** P<0.01. FIG. 5Cprovides a graph of NV area over total retinal area quantitativelyassessed for isolectin stained retinal flat mounts prepared at P17 fromWT and Gcn2 knockout (Gcn2^(−/−)) neonates exposed to hyperoxia fromP7-P12 to induce OIR (N≧12). Data are means+/−SEM; **** P<0.0001. FIG.5D provides a graph of NV area over total retinal area quantitativelyassessed for isolectin stained retinal flat mounts prepared at P17 fromWT neonates exposed to hyperoxia from P7-P12 to induce OIR followed byintraocular injections of the indicated siRNAs targeting VEGF-A, Ido1,CHOP, or the corresponding non-target control (siNtc). Data aremeans+/−SEM; ****, P<0.0001; ***, P<0.001; **, P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Indoleamine 2,3-dioxygenase 1 (IDO1) is an extrahepatic enzyme thatcatabolizes the essential amino acid tryptophan independent of metabolicprocessing of tryptophan in the liver. Detection of elevated levels oftryptophan catabolites in the urine of bladder cancer patients was firstreported in the 1950's (Boyland et al. (1956) Biochem. J., 64:578-582;Boyland et al. (1955) Process. Biochem., 60:v). The determination thatcancer-associated tryptophan catabolism was not attributable to elevatedactivity of TDO2, the liver enzyme responsible for maintainingtryptophan homeostasis (Gailani et al. (1973) Cancer Res.,33:1071-1077), led, in part, to the discovery of IDO1 from rabbitintestine (Higuchi et al. (1967) Arch. Biochem. Biophys., 120:397-403;Higuchi et al. (1963) Federation Proc., 22:243). The inflammatorycytokine IFNγ was found to be a major inducer of IDO1 (Yoshida et al.(1981) Proc. Natl. Acad. Sci., 78:129-132), and a general consensusinitially formed around the idea of IDO1 elevation being a tumoricidaleffect of IFNγ exposure that starves the rapidly growing tumor cells ofthis essential amino acid (Ozaki et al. (1988) Proc. Natl. Acad. Sci.,85:1242-1246). More recently, however, a major conceptual shiftregarding IDO1 as a regulator of immune function has emerged from thefinding that IDO1 activity could suppress cytotoxic T cell activation(Mellor et al. (1999) Immunol. Today 20:469-473; Munn et al. (1999) J.Exp. Med., 189:1363-1372). The demonstration that administering thebioactive IDO1 pathway inhibitor 1-methyl-tryptophan (1MT) to pregnantmice could elicit T cell-dependent rejection of allogeneic mouseconcepti (Munn et al. (1998) Science 281:1191-1193; Mellor et al. (2001)Nat. Immunol., 2:64-68) dramatically cemented the concept of IDO1 as animmunosuppressive actor and sparked the idea that tumors mightappropriate this mechanism of protecting the ‘foreign’ fetus to overcomeimmunosurveillance. Experimental support for this concept has come fromstudies in a mouse skin epithelial tumor isograft model linking loss ofthe tumor suppressor gene Bin1 to IDO1 dysregulation and immune escape(Muller et al. (2005) Nat. Med., 11:312-319).

The historical focus in cancer research on oncogenes and tumorsuppressor genes is based on the core assumption that tumorigenesis isessentially a cell autonomous process. IDO1, however, falls outside thisconventional framework in that it can contribute to tumor developmentnot only when it is expressed within tumor cells where immunoediting maybe at play but also when it is expressed within the normal stroma.Specifically, its induction in antigen presenting cells, in particulardendritic cells and macrophages, has been implicated in promoting immunetolerance by suppressing effector T cells, converting naïve T cells toFoxP3⁺ Tregs and elevating the suppressive activity of “natural” Tregs(Munn et al. (2013) Trends Immunol., 34:137-143). Extratumoral inductionof IDO1 was first reported in the B16 melanoma tumor graft model inwhich no IDO1 was detectable in the tumors but was elevated in the tumordraining lymph nodes (TDLN) where it was localized to a specific subsetof dendritic cells (Munn et al. (2004) J. Clin. Invest., 114:280-290).Several different IDO1 inhibitory compounds have since been identifiedthat can produce highly significant B16 tumor growth suppression that isdependent both on intact T cell immunity and IDO1 function in the animal(Banerjee et al. (2008) Oncogene 27:2851-2857; Kumar et al. (2008) J.Med. Chem., 51:1706-1718; Muller et al. (2010) Cancer Res.,70:1845-1853). In the classical two stage skin carcinogenesis, anautochthonous tumor model not involving the inherently artificialengrafting of tumor cells, it has been shown that genetic loss of IDO1rendered mice resistant to papilloma development (Muller et al. (2008)Proc. Natl. Acad. Sci., 105:17073-17078). No evidence of IDO1 was foundin the developing lesions but rather again in the TDLN (Muller et al.(2008) Proc. Natl. Acad. Sci., 105:17073-17078). In this context wheretumor initiation and promotion are distinctly separable, IDO1 in theTDLN was found to be elevated in the tumor-promoting inflammatoryenvironment irrespective of tumor initiation.

Whereas IDO1 elevation in tumor cells may be selected as an escapemechanism during immunoediting, skin carcinogenesis data suggested thatelevated extratumoral IDO1 expression preceding tumor initiation candirect the immune environment to be more pro-tumorigenic. To test thisidea further, the development of tumors in the lungs was studied as thisis an organ in which the constitutive level of IDO1 is relatively high(Takikawa et al. (1986) J. Biol. Chem., 261:3648-3653). Genetic loss ofIDO1 resulted in a marked decrease in the pulmonary tumor burden in botha transgenic mouse model of de novo lung carcinoma as well as in anorthotopic graft model of metastatic breast cancer. In both instancesthis translated to a significant survival benefit in the Ido1^(−/−)animals (Smith et al. (2012) Cancer Discov., 2:722-735). Unexpectedly,vascularization in the lung was significantly reduced with the loss ofIDO1 (Smith et al. (2012) Cancer Discov., 2:722-735). This resultsuggests the possibility that IDO1 may have a role in supportingangiogenesis. This would be consistent with its physiological role inmaintaining pregnancy, as the inflammatory environment elicited byimplantation in the endometrium directs angiogenesis as well as immunetolerance (Holtan et al. (2011) Front. Biosci., 3:1533-1540).

Angiogenesis is critical to tumor development (Cao et al. (2011) Sci.Transl. Med., 3(114):114rv113). Unlike physiologic angiogenesis which istightly regulated, in cancer there is excessive and disorganized growthof blood vessels much like that induced by ischemia in tissues such asthe retina and lungs. In experimental models of ischemia, immune cellshave been shown to be important for pruning the excess vasculature andlimiting neovascularization (Ishida et al. (2003) Nat. Med., 9:781-788;Wagner et al. (2008) Am. J. Physiol. Lung Cell. Mol. Physiol.,294:L351-357), suggesting that immunity might play an importantanti-angiogenic role in tumors as well. In particular, IFNγ, aninflammatory cytokine, long recognized as a major inducer of IDO1, hasbeen shown to exert angiostatic activity against developing tumors.Indeed, angiostasis elicited by IFNγ, rather than direct tumor cellkilling, has been implicated as being the primary mechanism for both CD4and CD8 T cell-mediated tumor rejection (Qin et al. (2000) Immunity12:677-686; Qin et al. (2003) Cancer Res., 63:4095-4100). In thiscontext, the finding that loss of IDO1 resulted in diminished pulmonaryvascularization (Smith et al. (2012) Cancer Discov., 2:722-735)suggested the hypothesis that IDO1 induced by IFNγ might be working atcross purposes to limit IFNγ-mediated angiostasis and that this might bean important factor accounting for the ability of IDO1 to promote tumoroutgrowth. In this same study (Smith et al. (2012) Cancer Discov.,2:722-735), IDO1 loss was associated with attenuated induction of thecytokine IL6 which is known to be pro-angiogenic and has been shown tobe important for ischemia-induced neovascularization in the lungs(McClintock et al. (2005) J. Appl. Physiol., 99:861-866). In a model ofpulmonary metastasis, ectopic expression of IL6 could overcome theresistance exhibited by Ido1^(−/−) mice, supporting the biologicalrelevance of the regulatory impact that IDO1 exerts on IL6 in a settingof pulmonary tumor outgrowth. These findings lead to a novel conceptualparadigm whereby elevated IDO in the host environment acts downstream ofIFNγ-mediated angiostasis and upstream of IL6-mediated angiogenesis fromthe very onset of tumor initiation to shift the immune response towardstumor promotion.

In order to directly test whether IDO1 supports pathologic angiogenesis,a mouse model of oxygen-induced retinopathy (OIR) (Connor et al. (2009)Nat. Protoc., 4:1565-1573) has been studied. As in cancer, inflammatorystimuli drive neovascularization in proliferative retinopathy and theOIR model has proved to be a temporally defined and quantifiablesurrogate system for studying the factors involved in tumor angiogenesis(Palmer et al. (2012) Br. J. Pharmacol., 165:1891-1903; Stahl et al.(2012) Blood 120:2925-2929). As shown herein, pathologicalneovascularization in the OIR model was significantly reduced in Ido1knockout mice (Ido1^(−/−)) mice or mice treated with inhibitors of IDO1or the IDO pathway (e.g., inhibitory nucleic acid molecules (e.g.,siRNA)), whereas normal retinal vascular development under normoxicconditions was unaffected in the Ido1^(−/−) mice. Likewise, thephysiological revascularization that occurs in response to ischemia wasnot impaired and even appears to have been enhanced as a result of IDO1loss as assessed by the relative sizes of the avascular areas. Thus,IDO1 inhibitors can overcome the indiscriminate targeting of normalcompensatory revascularization that is a detrimental consequence ofcurrent anti-VEGF antibody therapy. Consistent with IDO1 supportingneovascularization primarily by counteracting the angiostatic activityof IFNγ, the concurrent elimination of IFNγ in double knockoutIFNγ^(−/−) Ido1^(−/−) mice reverted the level of neovascularization inthe OIR model back to wild type levels. Conversely, IL6^(−/−) miceexhibited a reduction in neovascularization similar to that observed inIdo1^(−/−) mice consistent with IDO1 acting upstream to potentiate theangiogenic activity of IL6.

The present invention provides compositions and methods for theinhibition, prevention, and/or treatment of ocular diseases. The presentinvention also provides compositions and methods for the inhibition,prevention, and/or treatment of abnormal neovascularization (e.g., inthe eye). The present invention also provides compositions and methodsfor increasing and/or promoting normal vascularization and/or vasculargrowth (e.g., in the eye). The methods comprise administering at leastone inhibitor of the induction or activity of tryptophan degradation orof the downstream pathways that respond to this process to a subject. Ina particular embodiment, the inhibitor is a small molecule inhibitor. Ina particular embodiment, the inhibitor is an inhibitory nucleic acidmolecule (e.g., antisense, siRNA, shRNA, etc.) or a vector encoding thesame. In a particular embodiment, the inhibitor is an antibody orantibody fragment immunologically specific for the protein to beinhibited (e.g., a neutralizing antibody). In a particular embodiment,the methods comprise the administration of an IDO1 and/or IDO2 (see,e.g., PCT/US07/69271) inhibitor. In a particular embodiment, the methodcomprises the administration of an IDO1 inhibitor.

The inhibitors of the instant invention may inhibit the induction and/oractivity of tryptophan degradation and/or of the downstream pathwaysthat respond to this process. In a particular embodiment, the inhibitorof the instant invention inhibits the expression, induction, and/oractivity of IDO1 and/or of the downstream pathways from IDO1. The IDO1inhibitors may directly interfere with IDO1 activity, inhibit inductionof IDO1, or act as an inhibitor of the activation of downstreamsignaling pathways. In a particular embodiment, the IDO1 inhibitor is anantibody immunologically specific for IDO1. Examples of IDO1 inhibitorsare provided, without limitation, in PCT/US2014/022680 (e.g., tricycliccompounds related to imidazoisoindoles; compounds of Formulas I-V),PCT/US2012/033245 (e.g., fused imidazole derivatives; compounds ofFormula I or II), PCT/US2010/054289 (e.g., imidazole derivatives;compounds of Formulas I-VIII), PCT/US2009/041609 (e.g., compounds ofFormulas I-VIII), PCT/US2008/57032 (e.g., napthoquinone derivatives;compounds of Formula I, II, or III), PCT/US2008/085167 (e.g., compoundsof Formulas I-XLIV), PCT/US2006/42137 (e.g., compounds of Formula I),PCT/US2004/005155 (e.g., phenyl-TH-DL-trp(3-(N-phenyl-thiohydantoin)-indole), propenyl-TH-DL-trp(3-(N-allyl-thiohydantoin)-indole), and methyl-TH-DL-trp(3-(N-methyl-thiohydantoin)-indole)), PCT/US2004/005154 (e.g., compoundsof Formula I or II), U.S. Pat. No. 7,705,022 (e.g., compounds of FormulaI), U.S. Pat. No. 8,008,281 (e.g., phenyl-TH-DL-trp(3-(N-phenyl-thiohydantoin)-indole), propenyl-TH-DL-trp(3-(N-allyl-thiohydantoin)-indole), and methyl-TH-DL-trp(3-(N-methyl-thiohydantoin)-indole)), U.S. Pat. No. 7,714,139 (e.g.,compounds of Formula I or II), U.S. Patent Application Publication No.20140066625 (e.g., fused imidazole derivatives; compounds of Formula Ior II), U.S. Patent Application Publication No. 20130177590 (e.g.,N-hydroxyamidinoheterocycles; compounds of Formulas U.S. PatentApplication Publication No. 20140023663 (e.g., 1,2,5-oxadiazoles;compounds of Formula I), U.S. Patent Application Publication No.20080146624 (e.g., amidines; compounds of Formulas I or II), U.S. PatentApplication Publication No. 20080119491 (e.g., amidinoheterocycles;compounds of Formulas I-IV), U.S. Patent Application Publication No.20080182882 (e.g., N-hydroxyamidinoheterocycles; compounds of FormulaI), U.S. Patent Application Publication No. 20080214546 (e.g.,N-hydroxyamidinoheterocycles; compounds of Formula I), U.S. PatentApplication Publication No. 20060258719 (compounds of Formula I),Banerjee et al. (2008) Oncogene 27:2851-2857 (e.g., brassininderivatives;), and Kumar et al. (2008) J. Med. Chem., 51:1706-1718(e.g., phenyl-imidazole-derivatives). All references are incorporated byreference herein, particularly for the IDO1 inhibitors provided therein.

Inhibitors of IDO1 expression include, without limitation, inhibitors ofJAK/STAT (e.g., JAK, STAT3, STAT1) (Du et al. (2000) J. InterferonCytokine Res., 20:133-142, Muller et al. (2005) Nature Med., 11:312-319;Yu et al. (2014) J. Immunol., 193:2574-2586), NFκB (Muller et al. (2005)Nature Med., 11:312-319; Muller et al. (2010) Cancer Res.,70:1845-1853), KIT (Balachandran et al. (2011) Nature Med.,17:1094-1100), MET (Rutella et al. (2006) Blood 108:218-227; Giannoni etal. (2014) Haematologica 99:1078-1087), RAS/RAF/MEK (Liu (2010) Blood115:3520-3530), aryl hydrocarbon receptor (AHR) (Bessede et al. (2014)Nature 511:184-190; Litzenburger et al. (2014) Oncotarget 5:1038-1051),or vascular endothelial growth factor receptor (VEGFR) (Marti et al.(2014) Mem Inst Oswaldo Cruz 109:70-79). In a particular embodiment, theinhibitor is not an inhibitor of VEGFR.

Inhibitors of IDO1 downstream signaling include, without limitation,inhibitors of GCN2 (Munn et al. (2005) Immunity 22:633-642; Muller(2008) Proc Nat Acad Sci., 105:17073-17078), C/EBP homologous protein 10(CHOP-10; also known as gadd153; herein referred to as CHOP; Munn et al.(2005) Immunity 22:633-642), activating-transcription factor 4 (ATF4;Munn et al. (2005) Immunity 22:633-642; Thevenot et al. (2014) Immunity41:389-401), or aryl hydrocarbon receptor (AHR) (Opitz et al. (2011)Nature 478:197-203; Litzenburger et al. (2014) Oncotarget 5:1038-1051);or activators of mammalian target of rapamycin (mTOR) or protein kinaseC (PKC)-Θ (Metz et al. (2012) Oncoimmunology 1:1460-1468). In aparticular embodiment, the inhibitor of IDO1 downstream signaling is aninhibitor of IL6 (e.g., an antibody immunologically specific for IL6).

In a particular embodiment, the IDO1 inhibitor is not ethyl pyruvate. Ina particular embodiment, the IDO1 inhibitor (e.g., inhibitor ofdownstream signaling pathway) is 1-methyl-tryptophan, particularly1-methyl-D-tryptophan or a racemic mix comprising the same. In aparticular embodiment, the IDO inhibitor is 1-methyl-tryptophan,epacadosat (Incyte), or GDC-0919 (NewLink Genetics/Genentech).

In a particular embodiment, the ocular disease is characterized byabnormal/aberrant vascularization. In a particular embodiment the oculardisease is characterized by intraocular neovascularization. Theintraocular neovascularization may be, without limitation,neovascularization of the optic disc, iris, retina, choroid, cornea,and/or vitreous humour. Examples of ocular diseases include, withoutlimitation, glaucoma, pannus, pterygium, macular edema, maculardegeneration (e.g., age-related macular degeneration), retinopathy(e.g., diabetic retinopathy, vascular retinopathy, retinopathy ofprematurity), diabetic retinal ischemia, diabetic macular edema, retinaldegeneration, retrolental fibroplasias, retinoblastoma, corneal graftneovascularization, central retinal vein occlusion, pathological myopia,ocular tumors, uveitis, inflammatory diseases of the eye, andproliferative vitreoretinopathy. In a particular embodiment, the oculardisease is selected from the group consisting of retinopathy (e.g.,retinopathy of prematurity, diabetic retinopathy (e.g., proliferativediabetic retinopathy)) and macular degeneration (e.g., wet maculardegeneration).

As used herein, the term “macular degeneration” refers to oculardiseases wherein the macula—a small and highly sensitive part of theretina responsible for detailed central vision—degenerates and/or losesfunctional activity. The degeneration and/or loss of functional activitymay be due to any reason including, without limitation, cell death orapoptosis, decreased cell proliferation, and/or loss of normalbiological function. Macular degeneration may be wet (exudative orneovascular) or dry (non-exudative, atrophic or non-neovascular). In aparticular embodiment, the instant invention encompasses the treatmentof wet macular degeneration. Wet macular degeneration is typicallycharacterized by the formation of new vessels to improve the delivery ofblood to oxygen deprived retinal tissue (although the new vesselstypically rupture, causing bleeding and damage to surrounding tissue).Examples of macular degeneration diseases include, without limitation,age-related macular degeneration and Sorsby fundus dystrophy. As usedherein, the term “diabetic retinopathy” refers to changes in the retinadue to microvascular (e.g., retinal and choroidal) changes associatedwith diabetes. Without being bound by theory, small blood vessels withinthe retina, which are particularly susceptible to poor blood glucosecontrol, are damaged due to long-term exposure to high levels of bloodsugar (hyperglycemia). Diabetic retinopathy may affect one or both eyes,typically both eyes. The term “diabetic retinopathy” encompasses mild,moderate, or severe non-proliferative (simple) diabetic retinopathy(NPDR) and proliferative diabetic retinopathy (PDR). In a particularembodiment, the instant invention encompasses the treatment ofproliferative diabetic retinopathy. Proliferative diabetic retinopathyis typically characterized by the formation of new vessels to improvethe delivery of blood to oxygen deprived retinal tissue.

As used herein, the term “retinopathy of prematurity”, which is alsoknown as Terry syndrome or retrolental fibroplasia, refers to abnormalblood vessel development in the retina of the eye that occurs in infantsthat are born prematurely. Retinopathy of prematurity is typicallycharacterized by fibrovascular proliferation and the growth of abnormalnew vessels.

Based on the data provided herein, it is clear that the functional lossof IDO1 dramatically resolves abnormal neovascularization but has nodiscernable negative impact on normal vascular development. Notably,IDO1 inhibitors can be small molecules, which can allow for theiradministration to the eye via eye drops. This is in contrast to currentanti-VEGF antibody therapies which require intravitreal injections.

Compositions comprising at least one inhibitor of the induction oractivity of tryptophan degradation or of the downstream pathways thatrespond to this process (e.g., an IDO1 inhibitor) are also encompassedby the instant invention. In a particular embodiment, the compositioncomprises at least one inhibitor and at least one pharmaceuticallyacceptable carrier. The composition may further comprise at least oneother therapeutic compound for the inhibition, treatment, and/orprevention of the ocular disease or disorder (see, e.g., hereinbelow).Alternatively, at least one other therapeutic compound may be containedwithin a separate composition(s) with at least one pharmaceuticallyacceptable carrier. The present invention also encompasses kitscomprising a first composition comprising at least one inhibitor and asecond composition comprising at least one other therapeutic compoundfor the inhibition, treatment, and/or prevention of the ocular diseaseor disorder. The first and second compositions may further comprise atleast one pharmaceutically acceptable carrier.

The compositions of the instant invention are useful for treating anocular disease in a subject, particularly an ocular diseasecharacterized by abnormal vascularization. A therapeutically effectiveamount of the composition may be administered to the subject. Thedosages, methods, and times of administration are readily determinableby persons skilled in the art, given the teachings provided herein.

The compositions of the present invention can be administered by anysuitable route. In a particular embodiment, the compositions areadministered directly to the site of treatment (e.g., site forinhibiting abnormal neovascularization). In a particular embodiment, thecompositions described herein are administered in any way suitable toeffectively achieve a desired therapeutic effect in the eye. Forexample, the compositions of the instant invention may be administeredlocally to the eye, such as by topical administration, injection, ordelivery by an implantable device. Methods of administration include,without limitation, topical, intraocular (including intravitreal),transdermal, oral, intravenous, subconjunctival, subretinal, orperitoneal routes of administration. The compositions of the instantinvention can be in any form applicable for ocular administration. Forexample, the compositions may be in the form of eye drops, sprays,creams, ointments, gels (e.g., hydrogels), lens, films, implants,solutions, suspensions, and colloidal systems (e.g. liposomes,emulsions, dendrimers, micelles etc.). The compositions may also bemodified to increase the residence time of the compounds in the eye,provide a sustained release of compounds, and/or avoid toxicity andincrease ocular tolerability.

In general, the pharmaceutically acceptable carrier of the compositionis selected from the group of diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. The compositions can includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; and additives such as detergents and solubilizingagents (e.g., polysorbate 80), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). In a particularembodiment, the carrier is an aqueous or saline carrier. Thecompositions can also be incorporated into particulate preparations ofpolymeric compounds such as polylactic acid, polyglycolic acid, etc., orinto liposomes or nanoparticles. Such compositions may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of components of a pharmaceutical composition of the presentinvention. See, e.g., Remington's Pharmaceutical Sciences, (MackPublishing Co., Easton, Pa.). The pharmaceutical composition of thepresent invention can be prepared, for example, in liquid form, or canbe in dried powder form (e.g., lyophilized) for reconstitution prior toadministration. In a particular embodiment, the composition is anaqueous formulation with a pH physiologically compatible with the eye(e.g., a pH in the range from about 5.5 to about 8, particularly fromabout 6.0 to about 7.5). In a particular embodiment, the composition isan aqueous formulation having isotonic and physiological characteristicssuitable for ocular administration.

The methods of the instant invention may further comprise monitoring theocular disease or disorder in the subject after administration of thecomposition(s) of the instant invention to monitor the efficacy of themethod. For example, the subject may undergo an appropriate eye exam todetermine the severity of the ocular disease or disorder (e.g., todetermine if the severity of the ocular disease or disorder haslessened).

The methods of the instant invention may further comprise theadministration of at least one other therapeutic method for thetreatment of the ocular disease or disorder and/or the administration ofat least one other therapeutic compound for the treatment of the oculardisease or disorder. Methods of treating ocular diseases, particularocular disease characterized by abnormal vascularization, may also betreated with one or more additional therapies including, withoutlimitation, laser therapy (e.g., laser photocoagulation and photodynamictherapy (e.g., administration of verteporfin and application of light ofthe correct wavelength to activate verteporfin and obliterate thevessels)) and cryotherapy (freezing). With regard to diabeticretinopathy, compounds and/or therapies which inhibit and/or treat theunderlying diabetic condition may also be used. For example, compounds(e.g., insulin, metformin, meglumine, sorbitol) and methods whichmaintain optimal blood sugar levels can be administered to the subject.

As stated hereinabove, the compositions and methods of the instantinvention may further comprise one or more other compounds of methodsthat treat an ocular disease. In a particular embodiment, the furthercompound modulates ocular vascularization. Examples of other therapeuticcompounds include, without limitation, corticosteroids (e.g.,triamcinolone (e.g., intravitreal triamcinolone acetonide)),angiogenesis inhibitors, anti-vascular endothelial growth factor (VEGF)antibodies or compounds (e.g., anti-VEGF-A antibodies, ranibizumab,bevacizumab, WO 2014/056923), anti-vascular endothelial growth factoraptamers (e.g., pegaptanib), vascular endothelial growth factorinhibitors (e.g., aflibercept), and anecortave acetate.

Definitions

The following definitions are provided to facilitate an understanding ofthe present invention:

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease. The treatmentof an ocular disorder herein may refer to curing, relieving, and/orpreventing the ocular disorder, the symptom of it, or the predispositiontowards it.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, excipient,auxiliary agent or vehicle with which an active agent of the presentinvention is administered. Pharmaceutically acceptable carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water or aqueous saline solutionsand aqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described, for example, in “Remington's PharmaceuticalSciences” by E. W. Martin.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition resulting in adecrease in the probability that the subject will develop the condition.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc.

As used herein, the terms “host,” “subject,” and “patient” refer to anyanimal, including mammals such as humans.

As used herein, the term “small molecule” refers to a substance orcompound that has a relatively low molecular weight (e.g., less than2,000). Typically, small molecules are organic, but are not proteins,polypeptides, or nucleic acids.

The phrase “small, interfering RNA (siRNA)” refers to a short (typicallyless than 30 nucleotides long, particularly 12-30 or 20-25 nucleotidesin length) double stranded RNA molecule. Typically, the siRNA modulatesthe expression of a gene to which the siRNA is targeted. Methods ofidentifying and synthesizing siRNA molecules are known in the art (see,e.g., Ausubel et al. (2006) Current Protocols in Molecular Biology, JohnWiley and Sons, Inc). As used herein, the term siRNA may include shorthairpin RNA molecules (shRNA). Typically, shRNA molecules consist ofshort complementary sequences separated by a small loop sequence whereinone of the sequences is complimentary to the gene target. shRNAmolecules are typically processed into an siRNA within the cell byendonucleases. Exemplary modifications to siRNA molecules are providedin U.S. Application Publication No. 20050032733. Expression vectors forthe expression of siRNA molecules preferably employ a strong promoterwhich may be constitutive or regulated. Such promoters are well known inthe art and include, but are not limited to, RNA polymerase IIpromoters, the T7 RNA polymerase promoter, and the RNA polymerase IIIpromoters U6 and H1 (see, e.g., Myslinski et al. (2001) Nucl. AcidsRes., 29:2502 09).

“Antisense nucleic acid molecules” or “antisense oligonucleotides”include nucleic acid molecules (e.g., single stranded molecules) whichare targeted (complementary) to a chosen sequence (e.g., to translationinitiation sites and/or splice sites) to inhibit the expression of aprotein of interest. Such antisense molecules are typically betweenabout 15 and about 50 nucleotides in length, more particularly betweenabout 15 and about 30 nucleotides, and often span the translationalstart site of mRNA molecules. Antisense constructs may also be generatedwhich contain the entire sequence of the target nucleic acid molecule inreverse orientation. Antisense oligonucleotides targeted to any knownnucleotide sequence can be prepared by oligonucleotide synthesisaccording to standard methods.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof, that binds to a specific antigen. Asused herein, antibody or antibody molecule contemplates intactimmunoglobulin molecules, immunologically active portions of animmunoglobulin molecule, and fusions of immunologically active portionsof an immunoglobulin molecule.

The antibody may be a naturally occurring antibody or may be a syntheticor modified antibody (e.g., a recombinantly generated antibody; achimeric antibody; a bispecific antibody; a humanized antibody; acamelid antibody; and the like). The antibody may comprise at least onepurification tag. In a particular embodiment, the framework antibody isan antibody fragment. Antibody fragments include, without limitation,immunoglobulin fragments including, without limitation: single domain(Dab; e.g., single variable light or heavy chain domain), Fab, Fab′,F(ab′)₂, and F(v); and fusions (e.g., via a linker) of theseimmunoglobulin fragments including, without limitation: scFv, scFv₂,scFv-Fc, minibody, diabody, triabody, and tetrabody. The antibody mayalso be a protein (e.g., a fusion protein) comprising at least oneantibody or antibody fragment.

The antibodies of the instant invention may be further modified. Forexample, the antibodies may be humanized. In a particular embodiment,the antibodies (or a portion thereof) are inserted into the backbone ofan antibody or antibody fragment construct. For example, the variablelight domain and/or variable heavy domain of the antibodies of theinstant invention may be inserted into another antibody construct.Methods for recombinantly producing antibodies are well-known in theart. Indeed, commercial vectors for certain antibody and antibodyfragment constructs are available.

The antibodies of the instant invention may also be conjugated/linked toother components. For example, the antibodies may be operably linked(e.g., covalently linked, optionally, through a linker) to at least onecell penetrating peptide, detectable agent, imaging agent, or contrastagent. The antibodies of the instant invention may also comprise atleast one purification tag (e.g., a His-tag). In a particularembodiment, the antibody is conjugated to a cell penetrating peptide.

As used herein, the term “immunologically specific” refers toproteins/polypeptides, particularly antibodies, that bind to one or moreepitopes of a protein or compound of interest, but which do notsubstantially recognize and bind other molecules in a sample containinga mixed population of antigenic biological molecules.

A “cell-penetrating peptide” refers to a peptide which can transduceanother peptide, protein, or nucleic acid into a cell in vitro and/or invivo—i.e., it facilitates the cellular uptake of molecules. Examples ofcell penetrating peptides include, without limitation, Tat peptides,penetratin, transportan, and the like.

The following examples are provided to illustrate various embodiments ofthe present invention. They are not intended to limit the invention inany way.

Example 1

Based on the observation that Ido1 knockout mice exhibit reduced bloodvessel density in the lungs, the impact of the genetic loss of IDO1 inan oxygen-induced retinopathy (OIR) model was evaluated in order todetermine if IDO1 has a role in supporting angiogenesis. The OIR modelis a reliable, temporally defined, and quantifiable system for studyingneovascularization over a period of 17 days. Like tumors, proliferativeretinopathy is characterized by excessive and disorganized growth ofpathologic blood vessels. Tumor angiogenesis studies have previouslyincorporated OIR as a surrogate model system.

The OIR is schematically presented in Connor et al. (Nat Protoc. (2009)4:1565-73; FIG. 1). Briefly, mice were placed in a Plexiglas® chamberconnected to an oxygen delivery system that provided a hyperoxicenvironment, which causes vessel regression in the central retina andcessation of normal radial vessel growth. Animals were kept in thehyperoxic environment in the chamber from postnatal (p) day 7 until P12and then returned to atmospheric levels of oxygen (21%). Animals weresacrificed at P17—the peak of neovascularization—and their eyes wereenucleated. The retinas were then extracted and labeled with FITC-lectinto fluorescently stain retinal vasculature and to quantitatively analyzeneovascularization.

Representative microscopic images of FITC-lectin stained retinal flatmounts from wild type (WT) and Ido1 knockout (Ido1^(−/−)) mice at P17are shown in FIG. 1A. It was readily apparent just from the images thatthe extent of neovascularization was markedly reduced in the eyes fromIdo1^(−/−) mice. Quantitative comparison between the two groups, shownin FIG. 1B (left panel), confirmed that there was a highly significantreduction in neovascularization in the mice lacking IDO1 (P<0.0001).Control animals maintained under normoxic conditions throughout theexperiment demonstrated no appreciable evidence of abnormalneovascularization and the normal vascular development that occurred wasindistinguishable between WT and Ido1^(−/−) animals. In contrast to thereduction in abnormal neovascularization that resulted from the loss ofIDO1, the significant (P=0.002) reduction in the avascular regionobserved in Ido1^(−/−) animals relative to their wild type counterparts,shown in FIG. 1B (right panel), is indicative of an improvement innormal vascular regrowth occurring in mice lacking IDO1.

IFNγ, an inflammatory cytokine, long recognized as a major inducer ofIDO1, has been shown to exert angiostatic activity against developingtumors, which was implicated in these studies as the primary mechanismfor both CD4 and CD8 T cell dependent tumor rejection. The loss of IDO1results in diminished pulmonary vascularization (Smith et al. (2012)Cancer Discovery 2:722-735), suggesting that IDO1 might be working atcross purposes to limit IFNγ-mediated angiostasis. In this same study,IDO1 loss was also associated with the attenuated induction of thepro-angiogenic inflammatory cytokine IL6, suggesting that IDO1 might beacting upstream of IL6 to promote angiogenesis. Consistent with IDO1supporting neovascularization primarily by counteracting the angiostaticactivity of IFNγ, the concurrent elimination of IFNγ in double knockoutIfng^(−/−) Ido1^(−/−) mice reverted the level of neovascularization inthe OIR model back to wild type levels (FIG. 2A). Conversely, Il6^(−/−)mice exhibited a reduction in neovascularization in the OIR modelsimilar to that observed in Ido1^(−/−) mice, which was likewise reversedby the concurrent elimination of IFNγ in double knockout Ifng^(−/−)Il6^(−/−) mice (FIG. 2B), consistent with IDO1 acting upstream topotentiate the angiogenic activity of IL6 as an important contributingfactor in IDO1's ability to support neovascularization.

Example 2

B4-Alexa Fluor® 488-isolectin stained retinal flat mounts were preparedat P17 from wild type (WT), Ido1 knockout (Ido1^(−/−)), and Ido2knockout (Ido2^(−/−)) neonates exposed to normoxia or to hyperoxia fromP7-P12 to induce oxygen-induced retinopathy (OIR). FIG. 3 providesrepresentative microscopic images of B4-Alexa Fluor® 488-isolectinstained retinal flat mounts from WT and Ido1^(−/−) mice at P17(Ido2^(−/−) images not shown). The left column provides images ofretinal flat mounts of mice maintained under normoxia conditions (N≧10).Both WT and Ido1^(−/−) mice show normal vasculature. The middle columnand right column (higher magnification) provide images of retinal flatmounts of mice exposed to hyperoxia conditions from P7-P12 to triggerOIR (N≧16). The difference in neovascular tufts between WT andIdo1^(−/−) mice is evident as the Ido1^(−/−) mice show significantlyless neovascularization. Neovascular (NV) area over total retinal areawas quantitatively assessed for each cohort and the data are presentedas a bar graph of the means±SEM in FIG. 4A. Significance was determinedbetween the different pairwise combinations by one-way ANOVA withTukey's multiple comparison test (***, P<0.001; ns, not significant). Asseen in FIG. 4A, neovasculature was only negatively impacted inIdo1^(−/−) mice, but not Ido2^(−/−) mice.

The attenuated OIR neovascularization associated with IDO1 loss wasdetermined to be independent of the strain of mouse. Briefly, B4-AlexaFluor® 488-isolectin stained retinal flat mounts were prepared at P17from WT and Ido1^(−/−) neonates of both the BALB/c and C57BL/6 strainbackgrounds that were exposed to hyperoxia from P7-P12 to induce OIR(N≧14). NV area over total retinal area was quantitatively assessed foreach cohort and the data are presented as a bar graph of the means±SEMin FIG. 4B. Significance between the indicated pairs was determined by2-tailed Student's t test (**** P<0.0001). Similar levels of attenuationin neovascularization are observed in both strains of mice.

Normal vascularization of the retina under normoxic conditions isunaffected by the loss of IDO1. B4-Alexa Fluor® 488-isolectin stainedretinal flat mounts were prepared at P17 from WT and Ido1^(−/−) neonatesmaintained under constant normoxia (N=10). Vessel area over totalretinal area was quantitatively assessed for each cohort and the dataare presented as a bar graph of the means±SEM in FIG. 5A. Significancebetween the pair was determined by 2-tailed Student's t test (ns, notsignificant).

As noted in Example 1, the concurrent elimination of interferon-γ (IFNγ)in double knockout Ifng^(−/−) Ido1^(−/−) mice reverted the level ofneovascularization in the OIR model back to wild type levels. This isconsistent with the model that IDO1 supports neovascularizationprimarily by counteracting the angiostatic activity of IFNγ. Here, it isshown that antibody-mediated IFNγ neutralization restores OIRneovascularization in mice lacking IDO1. B4-Alexa Fluor® 488-isolectinstained retinal flat mounts were prepared at P17 from Ido1^(−/−)neonates exposed to hyperoxia from P7-P12 to induce OIR followed byintraocular injections of IFNγ neutralizing antibody or an isotypematched negative control antibody (N≧9). NV area over total retinal areawas quantitatively assessed for each cohort and the data are presentedas a bar graph of the means±SEM in FIG. 5B. Significance between theindicated pair was determined by 2-tailed Student's t test (**, P<0.01).

GCN2 is part of the IDO1 downstream signaling pathway. Herein, it isshown that the loss of GCN2 (through use of a GCN2 knockout mouse)results in attenuated OIR neovascularization. B4-Alexa Fluor®488-isolectin stained retinal flat mounts were prepared at P17 from WTand Gcn2^(−/−) neonates exposed to hyperoxia from P7-P12 to induce OIR(N≧12). NV area over total retinal area was quantitatively assessed foreach cohort, and the data are presented as a bar graph of the means±SEMin FIG. 5C. Significance between the pair was determined by 2-tailedStudent's t test (****, P<0.0001). These results demonstrate thatremoving a member of the IDO1 downstream signaling pathway produces asimilar effect as the loss of IDO1 itself. As such, it is clear thatinhibitors of members of the IDO1 downstream pathway may be used toachieve the same effect as IDO1 inhibitors.

OIR neovascularization was also attenuated by siRNA mediated knock-downof vascular endothelial growth factor A (VEGF-A), Ido1, and C/EBPhomologous protein 10 (CHOP-10; also known as gadd153; herein referredto as CHOP), which is an IDO1 downstream signaling gene. B4-Alexa Fluor®488-isolectin stained retinal flat mounts were prepared at P17 from WTneonates exposed to hyperoxia from P7-P12 to induce OIR followed byintraocular injections of an siRNA targeting VEGF-A, Ido1, CHOP, or acorresponding non-target control (Ntc). NV area over total retinal areawas quantitatively assessed for each cohort, and the data are presentedas a bar graph of the means±SEM in FIG. 5D. Significance between theindicated pairs was determined by 2-tailed Student t test (****,P<0.0001; ***, P<0.001; **, P<0.01). The reduction in IDO1 was confirmedwith anti-IDO1 antibody staining of retinal flat mounts. The use ofsiRNA demonstrates that administration into the eye of an agent thattargets IDO1 also attenuate neovascularization, as observed with thegenetic knockouts. The observed effect is at least as robust if notbetter than targeting VEGF-A. Targeting CHOP demonstrates that removinganother component of the IDO1 downstream signaling pathway produces asimilar effect as the direct loss of IDO1.

Several publications and patent documents are cited in the foregoingspecification in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A method for treating an ocular disease in asubject in need thereof, said method comprising the administration of atleast one inhibitor of tryptophan catabolism to the eye of said subject.2. The method of claim 1 comprising the administration of a compositioncomprising at least one inhibitor of indoleamine 2,3-dioxygenase-1(IDO1) and at least one pharmaceutically acceptable carrier.
 3. Themethod of claim 2, wherein said IDO1 inhibitor is an inhibitor of IDO1downstream signaling.
 4. The method of claim 2, wherein said IDO1inhibitor is 1-methyl-trypophan.
 5. The method of claim 1, wherein saidocular disease is characterized by abnormal vascularization.
 6. Themethod of claim 5, wherein said ocular disease is a retinopathy.
 7. Themethod of claim 5, wherein said ocular disease is retinopathy ofprematurity.
 8. The method of claim 5, wherein said ocular disease iswet macular degeneration.
 9. The method of claim 3, wherein saidinhibitor of IDO1 downstream signaling is an inhibitor of GCN2, C/EBPhomologous protein 10 (CHOP-10), aryl hydrocarbon receptor (AHR), oractivating-transcription factor 4 (ATF4).
 10. The method of claim 2,wherein said IDO1 inhibitor is a small molecule inhibitor.
 11. Themethod of claim 2, wherein said IDO1 inhibitor is an inhibitory nucleicacid molecule.
 12. The method of claim 11, wherein said inhibitorynucleic acid molecule is an antisense molecule or an siRNA.
 13. Themethod of claim 2, wherein said IDO1 inhibitor is an antibody orfragment thereof.
 14. The method of claim 2, wherein said composition isadministered topically to the eye.
 15. The method of claim 2, whereinsaid composition is administered by intravitreal injection.
 16. Acomposition comprising at least one inhibitor of indoleamine2,3-dioxygenase-1 (IDO1) and at least one pharmaceutically acceptablecarrier for topical administration to the eye of a subject.
 17. Thecomposition of claim 16 formulated as an eye drop.